AEM Accepts, published online ahead of print on 24 October 2014 Appl. Environ. Microbiol. doi:10.1128/AEM.02573-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Salivary mucins protect surfaces from colonization by cariogenic bacteria
2 3
Authors and Affiliations
4
Erica Shapiro Frenkel1, Katharina Ribbeck2*
5 6
1
7
MA, 02139
8
2
9
02139
[email protected], Biological Sciences in Dental Medicine, Harvard University, Cambridge,
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA,
10 11
*Correspondence
12
[email protected]
13 14
Running Title
15
Mucins protect surfaces from S. mutans colonization
16 17 18
Abstract:
19
Understanding how the body’s natural defenses function to protect the oral cavity from the
20
myriad of bacteria that colonize its surfaces is an ongoing topic of research that can lead to
21
breakthroughs in treatment and prevention. One key defense mechanism on all moist epithelial
22
linings, such as the mouth, gastrointestinal tract and lungs, is a layer of thick, well-hydrated
23
mucus. The main gel-forming component of mucus are mucins, large glycoproteins that play a
24
key role in host defense. This study focuses on elucidating the connection between MUC5B
25
salivary mucins and dental caries, one of the most common oral diseases. Dental caries are
26
predominantly caused by Streptococcus mutans adherence and biofilm formation on the tooth
27
surface. Once S. mutans adheres to the tooth, it produces organic acids as metabolic
28
byproducts that dissolve tooth enamel, leading to cavity formation. We utilize colony forming
29
units and fluorescent microscopy to quantitatively show that S. mutans attachment and biofilm
30
formation is most robust in the presence of sucrose and that aqueous solutions of purified
31
human MUC5B protect surfaces by acting as an anti-biofouling agent in the presence of
32
sucrose. In addition, we find that MUC5B does not alter S. mutans growth and decreases
33
surface attachment and biofilm formation by maintaining S. mutans in the planktonic form.
34
These insights point to the importance of salivary mucins in oral health and lead to a better
35
understanding of how MUC5B could play a role in cavity prevention or diagnosis.
36 37 38 39
Introduction: One of the body’s key defense mechanisms on wet epithelial linings, such as the mouth,
40
gastrointestinal tract and lungs, is a layer of thick, well-hydrated mucus. The viscoelastic
41
properties of mucus are attributed to mucins, large glycoproteins that play a key role in host
42
defense and maintaining a healthy microbial environment1–3. Defects in mucin production can
43
lead to diseases such as ulcerative colitis when mucins are under produced or cystic fibrosis
44
and asthma when mucins are overproduced4–6. In addition, studies have shown that mucins can
45
interact with microbes such as H. pylori, H. parainfluenzae and Human Immunodeficiency Virus
46
(HIV)7–10. These diseases and microbial interactions highlight the necessity of mucins as one of
47
the body’s key natural defenses, however, few studies have focused specifically on the
48
connection between MUC5B salivary mucins and oral diseases. This study fills this gap in
49
understanding by exploring the connection between purified human MUC5B and the virulence of
50
Streptococcus mutans, one of the main cavity-causing bacteria naturally found in the oral
51
cavity11. MUC7 is another salivary mucin found in the oral cavity, but MUC5B is the primary
52
mucin component of the dental pellicle coating the soft and hard tissues in the oral cavity12,13.
53
Importantly, the effects of MUC5B are characterized in a clinically relevant 3D model that
54
mimics the natural environment in the oral cavity; mucins are secreted into an aqueous phase
55
as opposed to a 2D surface coating, which can create artificially concentrated amounts of
56
surface mucins14–16. Suspending MUC5B in media allows polymer domains to interact
57
preventing collapse of the hydrogel structure8,17,18.
58
Understanding the structure of MUC5B illustrates how this specific glycoprotein can play
59
such a dominant role in maintaining oral health. There are several serotypes of mucins
60
throughout the body, but MUC5B is the predominant polymeric mucin found in the oral cavity
61
and female genital tract19,20. In the oral cavity, MUC5B is produced by goblet cells in the
62
submandibular and sublingual glands2. The peptide backbone is composed of a Variable
63
Number of Tandem Repeat (VNTR) section that has repeating sequences rich in serine,
64
threonine and proline, which participate in O-glycosylation1, 2,21. Because of the extended VNTR
65
region, MUC5B is composed of approximately 80% carbohydrate in the form of O-linked glycan
66
chains and 20% protein, consisting of the peptide backbone22,23. MUC5B’s complex structure
67
allows it to interact with an array of different salivary proteins and microbes to maintain a healthy
68
oral cavity24, 25. The exact mechanisms through which MUC5B provides defense are not well
69
understood, but it has been proposed that it acts as a physical protective barrier, provides
70
lubrication and has antimicrobial properties13,25,26.
71
S. mutans is a biofilm-forming facultative anaerobic bacteria that produces three
72
glucosyltransferase enzymes to synthesize glucans from dietary sugar27–29. Glucans are sticky
73
polymers that allow the bacteria to attach to the tooth surface and form an extracellular matrix
74
that protects it from host defenses and mechanical removal30,31. Once the bacteria attach to the
75
tooth surface, organic acids, which are produced as metabolic byproducts, become
76
concentrated within the extracellular matrix and cause a drop in pH from neutral to 5 or below.
77
This acidic environment begins dissolving tooth enamel leading to cavity formation, and S.
78
mutans’ high tolerance for acidic environments gives it an ecological advantage. Without proper
79
hygiene and nutritional awareness, S. mutans can proliferate quickly causing serious damage to
80
tooth structure. S. mutans biofilm formation is particularly problematic in the interproximal
81
spaces between teeth where mechanical removal is difficult.
82 83
Because S. mutans attachment and biofilm formation are critical steps in cavity formation, we use colony forming units (CFU) and fluorescence microscopy to quantify the
84
effects of supplemental sugar and purified human salivary MUC5B on these key stages of
85
disease progression. We first validate our mucin studies by showing that S. mutans attachment
86
and biofilm formation is most robust in the presence of sucrose as opposed to glucose. When
87
supplemental MUC5B is added in the presence of sucrose, however, S. mutans attachment and
88
biofilm formation are significantly decreased. Although the number of surface attached bacteria
89
decrease in the presence of MUC5B, we show that bacterial growth is unchanged in the
90
presence of MUC5B and the observed effects are due to increased S. mutans in the planktonic
91
form. These findings that link MUC5B with S. mutans virulence could significantly impact our
92
understanding of the pathogenesis of cavity formation and aid in the development of novel oral
93
diagnostic methods or strategies for disease prevention.
94 95 96
Methods:
97
Bacterial strains and growth conditions. The bacterial strain Streptococcus mutans UA159
98
was kindly given as a gift by Dr. Dan Smith (Forsyth Institute). For sucrose and glucose
99
experiments, bacteria were grown overnight in Brain Heart Infusion media (BHI; Becton
100
Dickinson and Company) containing 1% sucrose (w/v) and BHI with 1% glucose (Sigma). For
101
experiments determining the effects of MUC5B, S. mutans was grown overnight in BHI with 1%
102
sucrose. BHI with 1% sucrose and either 0.3% MUC5B or methylcellulose (w/v, Sigma) were
103
used to resuspend the bacteria before inoculating into the experiment. Hydroxyapatite discs
104
(Clarkson Chromatography, Inc.) or glass chambered slides (LabTek) surfaces were used to
105
test S. mutans attachment and biofilm formation. Bacteria were grown and incubated at 37 °C
106
with 5% CO2.
107 108
Saliva collection. Submandibular saliva was collected from ten volunteers using a custom
109
vacuum pump set up. Specifically, two holes were cut into the cap of a 50 mL conical tube
110
(Falcon); the vacuum line was inserted into one hole and a small diameter Tygon collection tube
111
was inserted into the other hole (Saint Gobain Performance Plastics). Cotton swabs were used
112
to absorb volunteers’ parotid gland secretions. The collection tube was used to suck up pooled
113
unstimulated submandibular gland secretions from under the tongue. The collection vessel was
114
kept on ice at all times. Saliva from volunteers was pooled before MUC5B purification.
115 116
MUC5B purification. Immediately after collection, saliva was diluted using 5.5 M sodium
117
chloride containing 0.04% sodium azide so the final concentration of sodium chloride was 0.16
118
M. The following antibacterial agents and protease inhibitors were then added at the given
119
concentrations: BenzamidineHCl (5 mM), dibromoacetophenone (1 mM),
120
phenylmethylsulfonylfluoride (1 mM), and ethylenediaminetetraacetic acid (5 mM, pH 7)
121
(Sigma). Mucins in saliva were solubilized overnight by gentle stirring at 4 °C. Saliva was then
122
centrifuged at 3800 g for 10 minutes in a swinging bucket centrifuge to remove cellular debris.
123
MUC5B was purified using a Bio-Rad NGC Fast Protein Liquid Chromatography system
124
equipped with an XK 50 column packed with Sepharose CL-2B resin (GE Healthcare Bio-
125
Sciences). Mucin containing fractions were identified using a Periodic Schiff Assay and UV280
126
FPLC analysis. Fractions were then combined, dialyzed and concentrated using an ultrafiltration
127
device, then lyophilized for storage at -80 °C.
128 129
Colony forming unit assay to evaluate S. mutans attachment and biofilm formation. To
130
test the effects of sucrose or glucose on S. mutans physiology, S. mutans was grown to mid-
131
exponential phase in BHI with 1% sucrose and BHI with 1% glucose then equal numbers of
132
bacteria (107) from each culture were seeded in triplicate into wells containing glass or
133
hydroxyapatite surfaces. For experiments testing the effect of MUC5B, S. mutans was grown to
134
mid-exponential phase in BHI with 1% sucrose then seeded in triplicate into wells containing
135
BHI with 1% sucrose and 0.3% MUC5B or control media as indicated. For all experiments,
136
attachment was evaluated at 20, 40, and 60 minutes and biofilm formation at 6, 18 and 24
137
hours. Attachment is defined as time points up to 1 hour because the doubling time of S.
138
mutans is approximately 1.5 hours. Biofilm formation is all time points after 1 hour. At the end of
139
the time point, the surface was washed with phosphate buffered saline (PBS) to remove non-
140
adherent bacteria, fresh PBS was added, then adherent cells were lifted using a sterile pipette
141
tip. Suspended bacteria were vigorously pipetted to individualize the cells. The suspension was
142
diluted (10-1 to 10-7) and plated on BHI agar. CFUs were counted after 24-36 hours of
143
incubation. Statistically significant differences were determined using the student’s t-test with
144
p
1
Salivary mucins protect surfaces from colonization by cariogenic bacteria
2 3
Authors and Affiliations
4
Erica Shapiro Frenkel1, Katharina Ribbeck2*
5 6
1
7
MA, 02139
8
2
9
02139
[email protected], Biological Sciences in Dental Medicine, Harvard University, Cambridge,
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA,
10 11
*Correspondence
12
[email protected]
13 14
Running Title
15
Mucins protect surfaces from S. mutans colonization
16 17 18
Abstract:
19
Understanding how the body’s natural defenses function to protect the oral cavity from the
20
myriad of bacteria that colonize its surfaces is an ongoing topic of research that can lead to
21
breakthroughs in treatment and prevention. One key defense mechanism on all moist epithelial
22
linings, such as the mouth, gastrointestinal tract and lungs, is a layer of thick, well-hydrated
23
mucus. The main gel-forming component of mucus are mucins, large glycoproteins that play a
24
key role in host defense. This study focuses on elucidating the connection between MUC5B
25
salivary mucins and dental caries, one of the most common oral diseases. Dental caries are
26
predominantly caused by Streptococcus mutans adherence and biofilm formation on the tooth
27
surface. Once S. mutans adheres to the tooth, it produces organic acids as metabolic
28
byproducts that dissolve tooth enamel, leading to cavity formation. We utilize colony forming
29
units and fluorescent microscopy to quantitatively show that S. mutans attachment and biofilm
30
formation is most robust in the presence of sucrose and that aqueous solutions of purified
31
human MUC5B protect surfaces by acting as an anti-biofouling agent in the presence of
32
sucrose. In addition, we find that MUC5B does not alter S. mutans growth and decreases
33
surface attachment and biofilm formation by maintaining S. mutans in the planktonic form.
34
These insights point to the importance of salivary mucins in oral health and lead to a better
35
understanding of how MUC5B could play a role in cavity prevention or diagnosis.
36 37 38 39
Introduction: One of the body’s key defense mechanisms on wet epithelial linings, such as the mouth,
40
gastrointestinal tract and lungs, is a layer of thick, well-hydrated mucus. The viscoelastic
41
properties of mucus are attributed to mucins, large glycoproteins that play a key role in host
42
defense and maintaining a healthy microbial environment1–3. Defects in mucin production can
43
lead to diseases such as ulcerative colitis when mucins are under produced or cystic fibrosis
44
and asthma when mucins are overproduced4–6. In addition, studies have shown that mucins can
45
interact with microbes such as H. pylori, H. parainfluenzae and Human Immunodeficiency Virus
46
(HIV)7–10. These diseases and microbial interactions highlight the necessity of mucins as one of
47
the body’s key natural defenses, however, few studies have focused specifically on the
48
connection between MUC5B salivary mucins and oral diseases. This study fills this gap in
49
understanding by exploring the connection between purified human MUC5B and the virulence of
50
Streptococcus mutans, one of the main cavity-causing bacteria naturally found in the oral
51
cavity11. MUC7 is another salivary mucin found in the oral cavity, but MUC5B is the primary
52
mucin component of the dental pellicle coating the soft and hard tissues in the oral cavity12,13.
53
Importantly, the effects of MUC5B are characterized in a clinically relevant 3D model that
54
mimics the natural environment in the oral cavity; mucins are secreted into an aqueous phase
55
as opposed to a 2D surface coating, which can create artificially concentrated amounts of
56
surface mucins14–16. Suspending MUC5B in media allows polymer domains to interact
57
preventing collapse of the hydrogel structure8,17,18.
58
Understanding the structure of MUC5B illustrates how this specific glycoprotein can play
59
such a dominant role in maintaining oral health. There are several serotypes of mucins
60
throughout the body, but MUC5B is the predominant polymeric mucin found in the oral cavity
61
and female genital tract19,20. In the oral cavity, MUC5B is produced by goblet cells in the
62
submandibular and sublingual glands2. The peptide backbone is composed of a Variable
63
Number of Tandem Repeat (VNTR) section that has repeating sequences rich in serine,
64
threonine and proline, which participate in O-glycosylation1, 2,21. Because of the extended VNTR
65
region, MUC5B is composed of approximately 80% carbohydrate in the form of O-linked glycan
66
chains and 20% protein, consisting of the peptide backbone22,23. MUC5B’s complex structure
67
allows it to interact with an array of different salivary proteins and microbes to maintain a healthy
68
oral cavity24, 25. The exact mechanisms through which MUC5B provides defense are not well
69
understood, but it has been proposed that it acts as a physical protective barrier, provides
70
lubrication and has antimicrobial properties13,25,26.
71
S. mutans is a biofilm-forming facultative anaerobic bacteria that produces three
72
glucosyltransferase enzymes to synthesize glucans from dietary sugar27–29. Glucans are sticky
73
polymers that allow the bacteria to attach to the tooth surface and form an extracellular matrix
74
that protects it from host defenses and mechanical removal30,31. Once the bacteria attach to the
75
tooth surface, organic acids, which are produced as metabolic byproducts, become
76
concentrated within the extracellular matrix and cause a drop in pH from neutral to 5 or below.
77
This acidic environment begins dissolving tooth enamel leading to cavity formation, and S.
78
mutans’ high tolerance for acidic environments gives it an ecological advantage. Without proper
79
hygiene and nutritional awareness, S. mutans can proliferate quickly causing serious damage to
80
tooth structure. S. mutans biofilm formation is particularly problematic in the interproximal
81
spaces between teeth where mechanical removal is difficult.
82 83
Because S. mutans attachment and biofilm formation are critical steps in cavity formation, we use colony forming units (CFU) and fluorescence microscopy to quantify the
84
effects of supplemental sugar and purified human salivary MUC5B on these key stages of
85
disease progression. We first validate our mucin studies by showing that S. mutans attachment
86
and biofilm formation is most robust in the presence of sucrose as opposed to glucose. When
87
supplemental MUC5B is added in the presence of sucrose, however, S. mutans attachment and
88
biofilm formation are significantly decreased. Although the number of surface attached bacteria
89
decrease in the presence of MUC5B, we show that bacterial growth is unchanged in the
90
presence of MUC5B and the observed effects are due to increased S. mutans in the planktonic
91
form. These findings that link MUC5B with S. mutans virulence could significantly impact our
92
understanding of the pathogenesis of cavity formation and aid in the development of novel oral
93
diagnostic methods or strategies for disease prevention.
94 95 96
Methods:
97
Bacterial strains and growth conditions. The bacterial strain Streptococcus mutans UA159
98
was kindly given as a gift by Dr. Dan Smith (Forsyth Institute). For sucrose and glucose
99
experiments, bacteria were grown overnight in Brain Heart Infusion media (BHI; Becton
100
Dickinson and Company) containing 1% sucrose (w/v) and BHI with 1% glucose (Sigma). For
101
experiments determining the effects of MUC5B, S. mutans was grown overnight in BHI with 1%
102
sucrose. BHI with 1% sucrose and either 0.3% MUC5B or methylcellulose (w/v, Sigma) were
103
used to resuspend the bacteria before inoculating into the experiment. Hydroxyapatite discs
104
(Clarkson Chromatography, Inc.) or glass chambered slides (LabTek) surfaces were used to
105
test S. mutans attachment and biofilm formation. Bacteria were grown and incubated at 37 °C
106
with 5% CO2.
107 108
Saliva collection. Submandibular saliva was collected from ten volunteers using a custom
109
vacuum pump set up. Specifically, two holes were cut into the cap of a 50 mL conical tube
110
(Falcon); the vacuum line was inserted into one hole and a small diameter Tygon collection tube
111
was inserted into the other hole (Saint Gobain Performance Plastics). Cotton swabs were used
112
to absorb volunteers’ parotid gland secretions. The collection tube was used to suck up pooled
113
unstimulated submandibular gland secretions from under the tongue. The collection vessel was
114
kept on ice at all times. Saliva from volunteers was pooled before MUC5B purification.
115 116
MUC5B purification. Immediately after collection, saliva was diluted using 5.5 M sodium
117
chloride containing 0.04% sodium azide so the final concentration of sodium chloride was 0.16
118
M. The following antibacterial agents and protease inhibitors were then added at the given
119
concentrations: BenzamidineHCl (5 mM), dibromoacetophenone (1 mM),
120
phenylmethylsulfonylfluoride (1 mM), and ethylenediaminetetraacetic acid (5 mM, pH 7)
121
(Sigma). Mucins in saliva were solubilized overnight by gentle stirring at 4 °C. Saliva was then
122
centrifuged at 3800 g for 10 minutes in a swinging bucket centrifuge to remove cellular debris.
123
MUC5B was purified using a Bio-Rad NGC Fast Protein Liquid Chromatography system
124
equipped with an XK 50 column packed with Sepharose CL-2B resin (GE Healthcare Bio-
125
Sciences). Mucin containing fractions were identified using a Periodic Schiff Assay and UV280
126
FPLC analysis. Fractions were then combined, dialyzed and concentrated using an ultrafiltration
127
device, then lyophilized for storage at -80 °C.
128 129
Colony forming unit assay to evaluate S. mutans attachment and biofilm formation. To
130
test the effects of sucrose or glucose on S. mutans physiology, S. mutans was grown to mid-
131
exponential phase in BHI with 1% sucrose and BHI with 1% glucose then equal numbers of
132
bacteria (107) from each culture were seeded in triplicate into wells containing glass or
133
hydroxyapatite surfaces. For experiments testing the effect of MUC5B, S. mutans was grown to
134
mid-exponential phase in BHI with 1% sucrose then seeded in triplicate into wells containing
135
BHI with 1% sucrose and 0.3% MUC5B or control media as indicated. For all experiments,
136
attachment was evaluated at 20, 40, and 60 minutes and biofilm formation at 6, 18 and 24
137
hours. Attachment is defined as time points up to 1 hour because the doubling time of S.
138
mutans is approximately 1.5 hours. Biofilm formation is all time points after 1 hour. At the end of
139
the time point, the surface was washed with phosphate buffered saline (PBS) to remove non-
140
adherent bacteria, fresh PBS was added, then adherent cells were lifted using a sterile pipette
141
tip. Suspended bacteria were vigorously pipetted to individualize the cells. The suspension was
142
diluted (10-1 to 10-7) and plated on BHI agar. CFUs were counted after 24-36 hours of
143
incubation. Statistically significant differences were determined using the student’s t-test with
144
p
